Nucleic Acid Marker Ladder for Estimating Mass

ABSTRACT

The invention relates to a nucleic acid marker ladder which is a restriction endonuclease digest, wherein a nucleic acid restriction endonuclease digest is a collection of nucleic acid fragments resulting from complete digestion of one or more nucleic acids by one or more restriction endonucleases; the restriction endonuclease digest contains at least 3 fragments; and the size of the fragments in base pairs is a multiple of an integer, wherein the integer is 10 or more.

FIELD OF THE INVENTION

The present invention is in the field of molecular biology andspecifically relates to the technique of gel electrophoresis of nucleicacid fragments.

BACKGROUND OF THE INVENTION

Gel electrophoresis of nucleic acid is a well known technique inmolecular biology. Nucleic acid molecules are separated on the basis ofsize (length or molecular weight), and conformation (linear vs. nickedcircles vs. covalently closed circles). For a given conformation,electrophoretic mobility is inversely related to size.

Conventional agarose gel electrophoresis is commonly used for theseparation of nucleic acid fragments within a practical resolution limitof 50 kbp (Cantor, C. R. and Schimmel, P. R. (1980) BiophysicalChemistry, Vol. III, pp. 1012-1036, Freeman, San Francisco; andManiatis, T. et al. (1982) Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). A methodcalled pulsed field gel electrophoresis (PFGE) has been developed toprovide separation of DNA molecules up to 2 Mbp (Schwartz, D. C. et al.(1983) Cold Spring Harbor Symp. Quant. Biol. 47:189-195; and Schwartz,D.C. and Cantor, C. R. (1984) Cell 37:67-75).

A number of mixtures of nucleic acid fragments (“ladders”) arecommercially available that can be used as markers for determining orestimating the sizes of nucleic acid molecules during gelelectrophoresis. One type of ladder is constructed by digesting plasmidsor bacteriophage with one or more restriction enzymes. The size of themarker fragments will depend upon the natural location of therestriction enzyme site-within the molecule to be digested and willproduce a quasi-random size distribution. For example digestion ofbacteriophage λ (lambda) with HindIII produces fragments of 23,130,9,416, 6557, 4361, 2322, 2027, 564, and 125 base pairs (bp) (See Cat.No. 5612SA, Life Technologies, Inc. 1992 catalogue, Gaithersburg, Md.,p. 318).

Alternatively, a ladder may comprise fragments which vary linearly withmolecular weight, i.e. adjacent bands may differ by about 1000 basepairs (e.g “1 Kb DNA Ladder”, See Cat. No. 5615SA, Life Technologies,Inc. 1992 catalogue, Gaithersburg, Md., p. 323), 100 base pairs (e.g“100 bp DNA Ladder”, See Cat. No. 5628SA, Life Technologies, Inc. 1992catalogue, Gaithersburg, Md., p. 322), or 123 bp (e.g “123 bp DNALadder”, See Cat. No. 5613SA, Life Technologies, Inc. 1992 catalogue,Gaithersburg, Md., p. 323). Some ladders have been constructed and soldthat are logarithmically spaced (“GenePrint™”, cat. no. DG1911, Promega,Madison, Wis.).

Nucleic acid is visualized in agarose gels following electrophoresis bystaining with the florescent dye ethidium bromide (Sharp et al. (1973)Biochemistry 12:3055). Ethidium bromide contains a planar group thatintercalates between nucleic acid bases. The fixed position of thisplanar group and its close proximity to the nucleic acid bases cause theethidium bromide bound to the nucleic acid to display an increasedfluorescent yield compared to that of ethidium bromide in free solution(See Sambrook et al. (1989) Molecular Cloning, 2nd edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., page 6.15). Themolecular mass of a nucleic acid fragment can be determined followingagarose gel electrophoresis and ethidium bromide staining by comparingthe intensity of the fluorescence of a fragment of unknown molecularmass with the intensity of a similarly sized fragment of known molecularmass.

SUMMARY OF THE INVENTION

In general, the present invention provides a nucleic acid marker ladder.More specifically, the present invention provides a nucleic acid markerladder consisting essentially of a restriction endonuclease digestwherein

(a) the nucleic acid restriction endonuclease digest is a collection ofnucleic acid fragments resulting from the complete digestion of one ormore nucleic acids by one or more restriction endonucleases;

(b) the restriction endonuclease digest contains at least 3 fragments;and

(c) the size of the fragments in base pairs is a multiple of an integer,wherein the integer is 10 or more.

The present invention also provides a nucleic acid marker kit comprisinga carrier means having in close confinement therein at least onecontainer means where a first container means contains theabove-described nucleic acid marker ladder.

The present invention also provides a method of preparing a nucleic acidmarker ladder comprising:

(a) generating at least two polymerase chain reaction (PCR) productswherein each product is generated from a template comprising arestriction endonuclease site and a primer comprising the restrictionendonuclease site in the template;

(b) joining the PCR products to produce one or more nucleic acidmolecules; and

(c) completely digesting the nucleic acid molecules with at least onerestriction endonuclease

wherein a nucleic acid marker ladder is produced wherein the laddercontains at least 3 fragments and the size of the fragments in basepairs is a multiple of an integer, wherein the integer is 10 or more.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A plasmid map of pML1 (Mass Ladder).

FIG. 2. Restriction enzyme digest of pML1. Lane 1. 100 bp ladder (SeeCat. No. 5628SA, Life Technologies, Inc. 1992 catalogue, Gaithersburg,Md., p. 322); Lanes 2, 4-6, 8-9. SspI digest of pML1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid marker ladder.

In one embodiment, the present invention relates to a nucleic acidmarker ladder consisting essentially of a restriction endonucleasedigest, wherein

(a) the nucleic acid restriction endonuclease digest is a collection ofnucleic acid fragments resulting from the complete digestion of one ormore nucleic acids by one or more restriction endonucleases;

(b) the restriction endonuclease digest contains at least 3 fragments;and

(c) the size of the fragments in base pairs is a multiple of an integer,wherein the integer is 10 or more. In one preferred embodiment theinteger is 10. In another preferred embodiment, the integer is 25. Inyet another preferred embodiment, the integer is 50. In a furtherembodiment, the integer is 100. In another preferred embodiment, thecollection of nucleic acid fragments results from digestion of a nucleicacid by one restriction endonuclease. In a further preferred embodiment,the of the fragments can be approximately a multiple of an integer. Forexample, the fragment's size can be 101, 201, 301, and 401 bp.

In another embodiment, the present invention relates to a nucleic acidmarker kit comprising a carrier means having in closeconfinement/therein at least one container means where a first containermeans contains the above-described nucleic acid marker ladder.

A restriction endonuclease (also restriction enzyme) is an enzyme thathas the capacity to recognize a specific base sequence (usually 4, 5, or6 base pairs in length) in a DNA molecule, and to cleave the DNAmolecule where this sequence appears. For example, EcoRI recognizes thebase sequence GAATTC/CTTAAG.

The DNA molecules produced by digestion with a restriction endonucleaseare referred to as restriction fragments. Any given genome, plasmid orphage may be digested by a particular restriction endonuclease into adiscrete set of restriction fragments.

The most commonly used analytical method (though not the only one) forfractionating double-stranded DNA molecules on the basis of size isagarose gel electrophoresis. The principle of this method is that DNAmolecules migrate through the gel as though it were a sieve that retardsthe movement of the largest molecules to the greatest extent and themovement of the smallest molecules to the least extent. Note that thesmaller the DNA fragment, the greater the mobility under electrophoresisin the agarose gel. The DNA fragments fractionated by agarose gelelectrophoresis can be visualized directly by a staining procedure ifthe number of fragments included in the pattern is small. Preferably,the nucleic acid-containing agarose gel is stained with ethidiumbromide.

As will be understood by those of skill in the art, the nucleic acidmolecules used to form the marker ladder are preferably any linear orcircular DNA which is cleavable by a restriction enzyme. For example,the nucleic acid may be chromosomes, plasmids, cosmids or viral nucleicacid. Preferably, the nucleic acid molecules are plasmid or viralmolecules and derivatives thereof. The nucleic acid present in theplasmid or viral molecule may include exogenous nucleic acid which hasbeen joined to produce the plasmid or viral molecule. In one preferredembodiment, the nucleic acid is DNA.

In another embodiment, the present invention relates to a method ofpreparing a nucleic acid marker ladder comprising:

(a) generating at least two polymerase chain reaction (PCR) productswherein each product is generated from a template comprising arestriction endonuclease site and a primer comprising the restrictionendonuclease site in the template;

(b) joining the PCR products to produce one or more nucleic acidmolecules; and

(c) completely digesting the nucleic acid molecules with at least onerestriction endonuclease

wherein a nucleic acid marker ladder is produced wherein the laddercontains at least 3 fragments and the size of the fragments in basepairs is a multiple of an integer, wherein the integer is 10 or more.

To construct a nucleic acid molecule which when digested with arestriction endonuclease produces the marker ladder of the presentinvention, the number of fragments in the restriction endonucleasedigest and the desired size of the fragments are selected. In onepreferred embodiment, the restriction endonuclease digest contains atleast three fragments. In another preferred embodiment, the restrictionendonuclease digest contains 3, 4, 6, 8 or 10 fragments. In a furtherpreferred embodiment, the restriction endonuclease digest contains 6fragments.

The size of the fragments in base pairs is preferably selected to be amultiple of an integer, wherein the integer is 10 or more. In onepreferred embodiment, the integer is 10, 25, 50, or 100. In another

Preferably, one of the fragments contains an origin of replication (forexample, ori) such that the nucleic acid molecule may autonomouslyreplicate within a cell. It is also preferable that one of the fragmentscontains a selectable or screenable marker. The origin of replicationand the marker may be present on the same fragment. Transformantscontaining this DNA fragment may be cultured and selected with aselection agent corresponding to the selectable marker.

The nucleic acid molecule is preferably constructed from polymerasechain reaction (PCR) products. The polymerase chain reaction provides amethod for selectively increasing the concentration of a nucleic acidmolecule having a particular sequence even when that molecule has notbeen previously purified and is present only as a single copy in aparticular sample. The method can be used to amplify single or doublestranded nucleic acid. Reviews of the polymerase chain reaction areprovided by Mullis, K. B., Cold Spring Harbor Symp. Quant. Biol.51:263-273 (1986); Saiki, R. K., et al., Bio/Technology 3:1007-1012(1985); Mullis, K. B., et al., Methods in Enzymology 55:350 (1987);Mullis, K., et al., U.S. Pat. No. 4,683,202; Erhlich, H., U.S. Pat. No.4,582,788; and Saiki, R., et al., U.S. Pat. No. 4,683,194.

In one preferred embodiment, a PCR product has the desired restrictionendonuclease site internally. In another preferred embodiment, a PCRproduct has the desired restriction endonuclease site at the rightand/or left end of the PCR product. In a further preferred embodiment,the PCR product has the desired restriction endonuclease site internallyand at the right or left end of the PCR product.

The PCR product is preferably generated from a naturally occurringtemplate. The template is preferably plasmid, phage, or plant, animal,or bacterial genomic DNA. The template preferably has an internalrestriction endonuclease site. The desired restriction endonuclease siteat the right or left end of the PCR product is preferably generated by arestriction endonuclease site in the PCR primer.

The spacing of the PCR priming sites and the naturally occurringrestriction sites is preferably arranged so that when the PCR productsare joined together, the nucleic acid molecule so formed when harvestedfrom E. coli can be cut with the desired restriction endonuclease toproduce the digest of the present invention.

For example, if a ladder of 6 fragments is preferred, three PCR productsmay be joined together. Product 1 would preferably contain joinable endsA and C, wherein C contains the desired restriction endonuclease site.Product 1 would also preferably contain the desired restrictionendonuclease site (B) internally.

Product 1 A B C

Product 2 would preferably contain joinable ends D and F, wherein Fcontains the desired restriction endonuclease site. Product 2 would alsopreferably contain the desired restriction endonuclease site (E)internally.

Product 2 D E F

Product 3 would preferably contain joinable ends G and I, wherein Icontains the desired restriction endonuclease site. Product 3 would alsopreferably contain the desired restriction endonuclease site (H)internally.

Product 3 G H I

The primer ends are constructed such that C is joinable to D, F isjoinable to G, and I is joinable to A. The primer ends A, C, D, F, G,and I are variable and depend upon the desired size of the fragmentspresent in the digest.

The PCR products can be joined together using ligase (preferably, E.coli DNA ligase, Cat. No. 8052SA, Life Technologies, Inc., Gaithersburg,Md.) to produce a nucleic acid molecule. The molecule is then digestedwith at least one restriction endonuclease wherein a nucleic acid markerladder is produced wherein the ladder contains at least 3 fragments andthe size of the fragments in base pairs is a multiple of an integer,wherein the integer is 10 or more. Preferably, one restrictionendonuclease is used. The six fragments produced from digestion of theabove-described molecule are B-C, C-E, E-F, F-H, H-I, and I-B.

In the examples that follow, DNA from three sources was joined togetherto form a nucleic acid which when cleaved with a restriction enzymeproduces a marker ladder of 6 fragments wherein the fragments aremultiples of 100.

The present invention is useful as a standard to be used duringelectrophoresis. A marker ladder wherein the size of the fragments is amultiple of an integer (preferably an integer of 10 or more) isextremely convenient and easy to use since one skilled in the art canquickly calculate the size of an unknown nucleic acid fragment.

However, the marker ladder of the present invention not only allows oneto size a nucleic acid but also to determine the mass of the nucleicacid. The molecular mass of a nucleic acid fragment can be determinedfollowing agarose gel electrophoresis and ethidium bromide staining bycomparing the intensity of the florescence of a fragment of unknownmolecular mass with the intensity of a similarly sized fragment of knownmolecular mass. The molecular mass is easily determined because all ofthe fragments derive from a single nucleic acid molecule.

The present invention is described in further detail in the followingnon-limiting examples.

Example I Construction of an SspI Marker Ladder

A marker ladder was constructed from three PCR products. Each PCRproduct has an Ssp I at its right end, and an internal Ssp I site(naturally occurring in the DNA template). The spacing of the primingsites and the naturally occurring restriction sites was arranged so thatwhen the three PCR fragments were annealed together (uracil bases in the5′ ends of the primers, treated with uracil DNA glycosylase (LifeTechnologies, Inc., Gaithersburg, Md.); 3′ ends anneal; U.S. Pat. Nos.5,137,814 and 5,229,283) to form a circular molecule, the plasmid soformed when harvested from E. coli can be cut (to completion) into sixSsp I fragments, all of which are multiples of 100 bp.

Three polymerase chain reactions were performed with each of thefollowing three sets of primers:

                     A         Spacer          pUC coord. 525pUC left: 5′ [auc uga ccu cau] [aat tta][cgg aag cat aaa gtg taa agc ct] 3′                       B′       Ssp I        pUC coord. 2600 pUC right: 5′ [agu cac agc uau][aat att] [gga aat gtg cgc gga acc cc] 3′                       B         Spacer         Ad2 coord. 32645Adeno2 left: 5′[aua gcu gug acu] [aat tta][cta gtg aat cca cag aaa cta gc] 3′                          C′       Ssp I          Ad2 coord. 34620 Adeno2 right: 5′[aca ucu gga cuu] [aat att] [aga cat att gat aag gtg gcg ag] 3′                      C         Spacer        SV40 coord. 1127SV40 Left: 5′ [aag ucc aga ugu] [aat tta][ggg aca gtt tgg caa ggt ttt ta] 3′                        A′       Ssp I          SV40 coord. 1702 SV40 right: 5′ [aug agg uca gau][aat att] [taa gcc ttt ttg atg ttc atc agg] 3′

Each 50 μl polymerase chain reaction contained 0.3 μM of each primer(reaction (a) contained the pUC primers; reaction (b) contained theAdeno2 primers; and reaction (c) contained the SV40 primers), 1 μlAmpliTaq (Perkin Elmer Cetus), 1 ng of template DNA (SV40 cut with KpnI; Adenovirus 2; or pUC19 cut with EcoRI (all DNAs from LifeTechnologies, Inc., Gaithersburg, Md.)) and 1×PCR buffer (50 mM KCl, 10mM Tris HCl pH 8.3, 1.5 mM MgCl₂, 5 mM 2-mercaptoethanol). Cyclingconditions were 94° 5 min; thirty cycles of 94° 30 sec, 55° 30 sec. 72°2 min; and hold at 0°.

UDG cloning was used to clone the PCR products. One μl of each PCRproduct was combined with 14 μl water, 2 μl 110×PCR buffer, and 1 μl=1unit UDG (Life Technologies, Inc., Gaithersburg, Md.), incubated 37° 30min, and transformed 1 μl into DH5 alpha cells (Life Technologies, Inc.,Gaithersburg, Md.). About 2500 colonies were obtained. Twelve colonieswere picked for minipreps. Of these twelve, eleven gave the expected 6Ssp I fragments of the expected size.

One of these plasmids was chosen and named “pML1”. This plasmid isexactly 4700 bp, and contains six Ssp I (blunt) fragments, of sizes2000, 1200, 800, 400, 200 and 100 bp. Thus, when 470 ng of this completedigest are applied to a gel, the six fragments contain 200, 120, 80, 40,20 and 10 ng of DNA.

Example 2 Construction of a NotI Marker Ladder

A marker ladder can be constructed from PCR products. The sizes of theresulting DNAs can be selected by positioning the PCR primersappropriately. In addition, the incorporation of restriction sites inthe primers, which are absent in the template molecules that areamplified during PCR, allows the ends of the PCR products to be cleavedwith cognate restriction enzymes. If multiple PCR product DNAs arejoined together and transformed into bacteria, and if one or more of thePCR products contains an origin of replication and a selection marker,the joined molecule can be recovered from the bacteria as a recombinantmolecule. Digestion of the molecule with the restriction enzyme whosesites have been placed in the primers yields the desired fragments. Byusing a variety of restriction enzyme sites to link PCR products, and adifferent restriction enzyme to cleave the resulting recombinantplasmid, multiple DNAs can be joined together in a particular order togive the desired product.

Using these principles, a marker plasmid can be constructed as follows.Three PCR products are synthesized. Terminal restriction sites allowjoining the fragments in a unique order and orientation. NotI sites willbe used to release the three desired fragments from the resultingplasmid. Spacers at the ends of the PCR products ensure efficientrestriction enzyme cutting.

PCR product I, 2026 bp from pUC:                 Nsp V  Not I     pUC19 coord. 637-656 Left primer:5 10 nt spacer/tt{circumflex over ( )}cgaa/gc{circumflex over( )}ggccgg/taa tga atc ggc caa cgc gc 3                BssH I  pUC 19 coord. 2622-2603 Right primer:5 10 nt spacer/g{circumflex over ( )}cgcgc/ga cgt cag gtg gca ctt ttc 3PCR product II, 1026 bp from SV40 DNA:                 Mlu I   Not I        SV40 coord. 108-129 Left primer:5 10 nt spacer/a{circumflex over ( )}cgcgt/gc{circumflex over( )}ggccgc/ggt tgc tga cta att gag atg c 3                BamH I   SV40 coord. 1093-1074 Right primer:5 10 nt spacer/g{circumflex over ( )}gatcc/gtg agg tga gcc tag gaa tg 3PCR product III, 526 bp from adenovirus 2:                  Bgl II  Not I      Ad2 coord. 1022-1041 Left primer:5′ 10 nt spacer/a{circumflex over ( )}gatct/gc{circumflex over( )}ggccgc/ggt ctt gtc att atc acc gg 3′                 Cla I     Ad2 coord. 1507-1488 Right primer: 5′10 nt spacer/at{circumflex over ( )}cgat/gtt gcc cag act cgt taa gc 3′

Each PCR product is digested with the two enzymes that cut at each end,i.e., I+Nsp V and BssH II, II with Mlu I and BamH I, and III with Bgl IIand Cla I. The three digested products are mixed and joined, and theproducts are digested with the six restriction enzymes prior totransformation into E. coli. The amplified fragments must be chosen tolack internal sites for these enzymes. The only clones that should beproduced by this process should be 3500 bp plasmids which can be cleavedwith Not I to give three fragments of 2000, 1000, and 500 bp.Electrophoresis of 350 ng of this digest will give three bandscontaining 200, 100, and 50 ng DNA, respectively.

All publications mentioned hereinabove are hereby incorporated in theirentirety by reference.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention and appended claims.

1. A nucleic acid marker ladder consisting essentially of a restrictionendonuclease digest, wherein (a) the nucleic acid restrictionendonuclease digest is a collection of nucleic acid fragments resultingfrom the complete digestion of one or more nucleic acids by one or morerestriction endonucleases; (b) the restriction endonuclease digestcontains at least 3 fragments; and (c) the size of the fragments in basepairs is a multiple of an integer, wherein the integer is 10 or more. 2.The nucleic acid marker ladder according to claim 1, wherein the integeris
 10. 3. The nucleic acid marker ladder according to claim 1, whereinthe integer is
 25. 4. The nucleic acid marker ladder according to claim1, wherein the integer is
 50. 5. The nucleic acid marker ladderaccording to claim 1, wherein the integer is
 100. 6. The nucleic acidmarker ladder according to claim 1, wherein the collection of nucleicacid fragments results from digestion of a nucleic acid by onerestriction endonuclease.
 7. A nucleic acid marker kit comprising acarrier means having in close confinement therein at least one containermeans where the first container means contains a nucleic acid markerladder consisting essentially of a restriction endonuclease digest,wherein (a) the nucleic acid restriction endonuclease digest is acollection of nucleic acid fragments resulting from the completedigestion of one or more nucleic acids by one or more restrictionendonucleases; (b) the restriction endonuclease digest contains at least3 fragments; and (c) the size of the fragments in base pairs is amultiple of an integer, wherein the integer is 10 or more.
 8. Thenucleic acid marker kit according to claim 7, wherein the integer is 10.9. The nucleic acid marker kit according to claim 7, wherein the integeris
 25. 10. The nucleic acid marker kit according to claim 7, wherein theinteger is
 50. 11. The nucleic acid marker kit according to claim 7,wherein the integer is
 100. 12. The nucleic acid marker kit according toclaim 7, wherein the collection of nucleic acid fragments results fromdigestion of a nucleic acid by one restriction endonuclease.
 13. Amethod of preparing a nucleic acid marker ladder comprising: (a)generating at least two polymerase chain reaction (PCR) products whereineach product is generated from a template comprising a restrictionendonuclease site and a primer comprising the restriction endonucleasesite in the template; (b) joining the PCR products to produce a nucleicacid molecule; and (c) completely digesting one or more nucleic acidmolecules with at least one restriction endonuclease wherein a nucleicacid marker ladder is produced wherein the ladder contains at least 3fragments and the size of the fragments in base pairs is a multiple ofan integer, wherein the integer is 10 or more.
 14. A method of using anucleic acid marker ladder to estimate the mass of a nucleic acidcomprising: (a) electrophoresing a known amount of the marker ladder ofclaim 1 and an unknown amount of said nucleic acid on an agarose gel;and (b) comparing the mass of said marker ladder with the mass of saidnucleic acid.